CN113429408A - Nitrogen-containing heterocyclic compound and preparation method, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention relates to a nitrogen-containing heterocyclic compound, a preparation method thereof, a pharmaceutical composition and application thereof. The structural formula of the nitrogen-containing heterocyclic compound is shown as follows:

wherein Y and Z are independently selected from-CH and-N; r¹And R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl. On one hand, the nitrogen-containing heterocyclic compound is combined with a tubulin colchicine binding site to promote microtubule depolymerization and inhibit tumor cell proliferation; on the other hand, the protein kinase can inhibit the kinase activity by combining with an ATP binding site of the protein kinase, thereby realizing multi-channel simultaneous inhibition of proliferation and survival of tumor cells. Compared with medicines aiming at tubulin such as paclitaxel and the like, the medicine has better water solubility, simpler structure, easy synthesis and good drug resistance.

Description

Nitrogen-containing heterocyclic compound and preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a nitrogen-containing heterocyclic compound, and a preparation method, a pharmaceutical composition and application thereof.

Background

The malignant tumor (cancer) has become one of the major public health problems seriously threatening the health of Chinese population, according to the latest statistical data, the death of the malignant tumor accounts for 23.91 percent of the total death causes of residents, the morbidity and mortality of the malignant tumor are in a continuously rising state in recent decades, the medical cost caused by the malignant tumor exceeds 2200 hundred million every year, and the prevention and control situation is severe. Although the research and development of the antitumor drug have succeeded to a certain extent, the survival rate of tumor patients is improved to a certain extent, and the life quality of the patients is also improved to a certain extent, the antitumor drug has more defects in clinical treatment, such as drug resistance, selectivity, toxic and side effects and the like. Obviously, the significance of developing novel effective antitumor drugs is great.

Microtubules are a component of cytoskeleton in eukaryotic cells, are highly dynamic structures composed of alpha-tubulin and beta-tubulin, have polymerization and depolymerization properties, and play important roles in cell mitosis, cell signaling, intracellular transport, angiogenesis and the like. Influencing microtubule polymerization and depolymerization by targeting microtubule dynamics has proven to be an effective, important anti-cancer strategy. There are 6 defined tubulin binding sites, 3 of which have been studied more extensivelyRespectively, a taxane binding site, a vinblastine binding site and a colchicine binding site. Currently, a number of tubulin-targeting drugs are clinically used for tumor therapy, such as paclitaxel (which promotes microtubule polymerization) and vinblastine (which inhibits microtubule polymerization). Although these drugs have become the first choice for various cancer treatments, the clinical applications of these drugs are still limited, mainly due to: 1) the toxicity is high, and the dosage use is limited; 2) the drug resistance of tumor cells is easily caused by the influence of efflux pump P-gp protein (P-glycoprotein) no matter taxol or vinblastine drugs; 3) poor water solubility and susceptibility to allergic reactions in vehicles, such as paclitaxel, necessitates prior treatment with steroids and antihistamines, adding to the patient's therapeutic and economic burden. In addition, paclitaxel and vinblastine compounds have complex structure (structural formula is shown respectively

) The synthesis and modification difficulties are great, so that the development difficulty and the cost of the medicine based on the compound are high. Therefore, the development of novel tubulin inhibitors is of great interest.

Protein kinases are important messengers of cell life activities, and can catalyze the transfer of a gamma-phosphate group at the end of Adenosine Triphosphate (ATP) onto a substrate, influence the structure and activity of the substrate, and transmit various intracellular and extracellular signals to appropriately respond to environmental stimuli. In most cases, this phosphorylation reaction occurs at a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of a protein kinase. Protein kinases are involved in numerous physiological regulatory processes including cell survival, proliferation, differentiation, apoptosis, metabolism, and the like. Pathological and pharmacological studies have shown that the dysfunction of protein kinases is closely related to many diseases, including tumors, autoimmune diseases, inflammatory reactions, central nervous system diseases, cardiovascular diseases and diabetes. Protein kinases have proven to be ideal targets for drug intervention during the last two or three decades, especially for the development of antineoplastic drugs. By 1 month 2021, 52 protein kinase inhibitors have been FDA approved, with 46 being indicated for tumors. Although protein kinases have become important targets for the development of anti-tumor drugs, similar to other anti-cancer therapies, anti-cancer targeted kinase therapies are also resistant. Therefore, the research and development efforts have been directed to how to reduce the drug resistance of antitumor drugs and improve the antitumor efficacy.

Disclosure of Invention

Therefore, it is necessary to provide a nitrogen-containing heterocyclic compound which can act on tubulin and protein kinase simultaneously, so as to realize multi-pathway inhibition of proliferation and survival of tumor cells, improve anti-tumor effect, and reduce drug resistance.

In addition, the application of the nitrogen-containing heterocyclic compound, a pharmaceutical composition and a preparation method of the nitrogen-containing heterocyclic compound are also needed to be provided.

A nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, wherein the nitrogen-containing heterocyclic compound has a structural formula as shown in the following:

wherein Y and Z are independently selected from-CH and-N;

R¹and R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl;

when said R is¹Or said R²When substituted aryl or substituted heteroaryl, the corresponding substituents are each independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mHeterocyclyl, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, O-aminomethyl of a heteroatomOne of acyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino and N-acylamino, and m is 0,1 or 2.

In one embodiment, R is¹Is selected from

One of (1);

wherein R is³、R⁵、R⁷And R⁸Each independently selected from one of hydrogen, alkyl, halogen, alkoxy, haloalkyl, cyano, amino, substituted amino and nitro;

R^4a、R^4band R⁶Each independently selected from one of hydrogen, alkyl, X-substituted alkyl, cycloalkyl, X-substituted cycloalkyl, heterocyclyl, X-substituted heterocyclyl, aryl, X-substituted aryl, heteroaryl, and X-substituted heteroaryl, wherein X is selected from at least one of alkyl, halogen, hydroxy, amino, mercapto, alkoxy, alkylamino, cycloalkyl, heterocyclyl, carboxy, and carboxylate;

or R^4aAnd R^4bForm a 4-to 8-membered ring M with the nitrogen atom to which it is attached₁Said M is₁Is a substituted heterocyclic group or an unsubstituted heterocyclic group, said M₁Containing at least one of N, O or S (O)_mAnd said M is₁The substituent(s) is selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

In one embodiment, R³And R⁵With NR^4aR^4bAnd OR⁶One of them is connected and combined to form 4-8 membered ring M₂Said M is₂Is a substituted heterocyclic group or an unsubstituted heterocyclic group, said M₂Containing at least one of N, O or S (O)_mAnd the substituent of the substituted heterocyclic group formed is at least one selected from the group consisting of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

In one embodiment, theR²Is selected from

One kind of (1).

In one embodiment, the nitrogen-containing heterocyclic compound is selected from one of the following structural formulas:

a pharmaceutical composition comprising the nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof.

A tubulin inhibitor or a protein kinase inhibitor comprising the nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof.

The nitrogen-containing heterocyclic compound or the pharmaceutically acceptable salt thereof is applied to the preparation of a medicament for treating cancer or the preparation of a protein kinase inhibitor or the preparation of a tubulin inhibitor or the preparation of a multi-target medicament.

In one embodiment, the medicament for treating cancer is a medicament for treating leukemia, a medicament for treating rectal cancer, a medicament for treating colon cancer, a medicament for treating lung cancer, a medicament for treating liver cancer, a medicament for treating ovarian cancer, a medicament for treating pancreatic cancer, a medicament for treating prostate cancer, a medicament for treating breast cancer or a medicament for treating cervical cancer;

the protein kinase is selected from at least one of BCR-ABL, FLT3, JNK, MEK5, STK16 and BMPR;

the multi-target drug has double inhibitory activities of tubulin and protein kinase.

A preparation method of a nitrogen-containing heterocyclic compound comprises the following steps:

mixing compound A, R¹B(OH)₂Reacting palladium catalyst with alkaline reagent to prepare a compound B, wherein the structural formula of the compound A is shown in the specification

The structural formula of the compound B is

Reacting the compound B with an acidic reagent to prepare a nitrogen-containing heterocyclic compound, wherein the structural formula of the nitrogen-containing heterocyclic compound is shown in the specification

Wherein Y and Z are respectively and independently selected from one of-CH and-N, R¹And R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl;

when said R is¹And said R²When it is substituted aryl or substituted heteroaryl, said R¹And said R²Each of the substituents of (A) is independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mHeterocyclyl, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino and N-acylamino radicals of hetero atomsOne of (1); m is 0,1 or 2.

In one embodiment, the method further comprises the step of preparing the compound A;

when Y and Z are both-CH, the step of preparing said compound A comprises:

reacting compound C with NaH and SEMCl to prepare compound D, wherein the structural formula of the compound C is shown in the specification

The structural formula of the compound D is

Subjecting said compound D, R²B(OH)₂Reacting a palladium catalyst and an alkaline reagent to prepare the compound A; alternatively, the first and second electrodes may be,

one of Y and Z is-CH and the other is-N, and the preparation method of the compound A comprises the following steps:

reacting the compound E with TMPZnCl. LiC to prepare a compound F, wherein the structural formulas of the compound E and the compound F are respectively shown in the specification

Subjecting said compounds F and R²COCl and CuCN.2LiC to prepare a compound G, wherein the structural formula of the compound G is shown in the specification

Reacting said compounds G and N₂H₄·H₂O、CH₃OH reaction to prepare a compound H, wherein the structural formula of the compound H is shown in the specification

Reacting the compound H with NaH and SEMCl to prepare the compound A.

On one hand, the nitrogen-containing heterocyclic compound is combined with a tubulin colchicine binding site to promote microtubule depolymerization and inhibit tumor cell proliferation; on the other hand, the protein kinase can inhibit the activity of kinase by combining with an ATP binding site of the protein kinase, thereby realizing the multi-channel simultaneous inhibition of the proliferation and survival of tumor cells, improving the antitumor activity and reducing the drug resistance. Compared with medicines aiming at tubulin, such as paclitaxel, the medicine has better water solubility, simpler structure and easy synthesis.

Drawings

FIG. 1 is a graph showing the results of experiments on the tubulin inhibitory activity of the nitrogen-containing heterocyclic compounds prepared in the examples.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description taken in conjunction with the accompanying drawings. The detailed description sets forth the preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In this context, Me denotes methyl, Et denotes ethyl, SEMCl denotes 2- (trimethylsilyl) ethoxymethyl chloride and SEM denotes- (trimethylsilyl) ethoxymethyl.

The term "alkyl" refers to a chain-like saturated hydrocarbon containing primary (normal) carbon atoms or secondary carbon atoms or tertiary carbon atoms or quaternary carbon atoms or combinations thereof, excluding cyclic saturated hydrocarbons. Phrases containing the term, e.g., "C₁～C₆Alkyl "refers to an alkyl group containing 1 to 6 carbon atoms, which may be independently at each occurrence C₁Alkyl radical, C₂Alkyl radical, C₃Alkyl radical, C₄Alkyl radical, C₅Alkyl or C₆An alkyl group. Likewise, alkenyl and alkynyl refer to chain unsaturated hydrocarbons.

As used herein, "a combination thereof" includes any suitable combination of the listed items as long as the object of the present invention can be achieved.

"aryl" refers to an aromatic hydrocarbon group derived by removing a hydrogen atom from the aromatic ring compound, and may be a monocyclic aryl group or a fused ring aryl group or a polycyclic aryl group, at least one of which is an aromatic ring system for polycyclic ring species.

"heteroaryl" means that at least one carbon atom is replaced by a heteroatom in the aryl group. The hetero atom may be an N atom, an O atom, an S atom, or the like. For example, "C₃～C₁₀Heteroaryl "refers to a heteroaryl group containing 3 to 10 carbon atoms, which at each occurrence may be independently C₃Heteroaryl group, C₄Heteroaryl group, C₅Heteroaryl group, C₆Heteroaryl group, C₇Heteroaryl or C₈A heteroaryl group. Suitable examples include, but are not limited to: furan, benzofuran, thiophene, benzothiophene, pyrrole, pyrazole, triazole, imidazole, oxazole, oxadiazole, thiazole, tetrazole, indole, carbazole, pyrroloimidazole, pyrrolopyrrole, thienopyrrole, thienothiophene, furopyrrole, furofuran, thienofuran, benzisoxazole, benzisothiazole, benzimidazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, quinoline, isoquinoline, phthalazine, quinoxaline, phenanthridine, primadine, quinazoline, and quinazolinone.

"Heterocyclyl" means that at least one carbon atom is replaced by a heteroatom, which may be a N atom, an O atom, an S atom, etc., which may be a saturated or partially unsaturated ring, in addition to cycloalkyl. Phrases containing the term, e.g., "C₄～C₉The "heterocyclic group" means a heterocyclic group containing 4 to 9 carbon atoms, and each occurrence may be independently C₄Heteroalkyl group, C₆Heteroalkyl group, C₇Heteroalkyl group, C₈Heteroalkyl radicals or C₉A heteroalkyl group. Suitable examples include, but are not limited to: dihydropyridinyl, tetrahydropyridinyl (piperidinyl), tetrahydrothienyl, thiooxidised tetrahydrothienyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, indolinyl.

"halogen" means F, Cl, Br or I.

"carboxyl" includes-COOH structure, not limited to methylcarboxyl, but may also be ethylcarboxyl, propylcarboxyl, etc.

"carboxylate group" means — C (═ O) OR.

"amino" means-NH₂。

"pharmaceutically acceptable salt" refers to a salt of any compound of the indicated structure with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One class of salts is the salts of the compounds of the present invention with acids. The acid suitable for forming the salt may be an organic acid, an inorganic acid, a natural amino acid or an unnatural amino acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another class of salts is that formed from the compounds of the present invention and bases suitable for forming salts include, but are not limited to: alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts, and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

Currently approved microtubule inhibitors for cancer therapy are targeted to either the taxane binding site or the vinblastine binding site, and no tubulin inhibitor targeted to the colchicine binding site has been approved for the market. The tubulin inhibitors clinically acting on the binding sites of taxane and vinblastine at present mainly have the following problems: (1) tubulin inhibitors represented by paclitaxel and vinblastine have complex structures, are difficult to synthesize and have high cost; (2) high toxicity, usually manifested as neurotoxicity and myelosuppressive toxicity; (3) the water solubility is poor, and a chemical cosolvent is required to be used for dissolving, so that anaphylactic reaction is easily caused; (4) is prone to multi-drug resistance, especially P-gp mediated resistance. Thus, the present invention provides a nitrogen-containing heterocyclic compound capable of acting on the colchicine binding site. The compound has the advantages of novel structure, easy synthesis, good water solubility and good drug resistance, and can overcome the defects of the traditional tubulin inhibitor.

In addition, the nitrogen-containing compound can be combined with a tubulin colchicine binding site to inhibit the proliferation of tumor cells, can act on protein kinase, and can inhibit the activity of the kinase by combining with an ATP binding site of the protein kinase, thereby realizing the multi-channel simultaneous inhibition of the proliferation and survival of the tumor cells and improving the antitumor activity.

Similar to other anticancer therapies, resistance can also develop with anticancer targeted kinase therapies. The drug resistance mechanism can be mainly divided into two categories, one category is from the target kinase, and the other category comprises overexpression and drug resistance mutation. The other type does not directly relate to the target itself, but rather achieves resistance through a bypass signaling pathway. The reasonable combination of small molecule kinase inhibitors with different target points has become one of the efforts to resist drug resistance and improve the benefit of targeted kinase therapy.

Given the very complex mechanisms of occurrence of tumors, disorders involving multiple signaling pathways and multiple physiological processes are involved. Therefore, compared with single-target antitumor drugs, the multi-target drugs can simultaneously act on multiple targets to generate synergistic interaction, so that the curative effect is improved, and the occurrence of drug resistance can be reduced to a certain extent. On the basis, the invention provides a nitrogen-containing heterocyclic compound which can simultaneously act on multiple targets of kinase and tubulin and has multiple anti-tumor effects.

Specifically, the nitrogen-containing heterocyclic compound of one embodiment has a structural formula as follows:

wherein Y and Z are independently selected from-CH and-N. Further, Y and Z are both-CH, or Y is-CH and Z is-N, or Y is-N and Z is-CH.

R¹And R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl. Preferably, R¹And R²Each independently selected from one of substituted aryl and substituted heteroaryl.

When R is¹Or R²When substituted aryl or substituted heteroaryl, the corresponding substituents are each independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mOne of heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonylamino group, N-sulfonylamino group, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group and N-acylamino group of hetero atom; m is 0,1 or 2.

Preferably, R¹Is selected from

One kind of (1).

Wherein R is³、R⁵、R⁷And R⁸Each independently selected from one of hydrogen, alkyl, halogen, alkoxy, haloalkyl, cyano, amino, substituted amino and hydroxyl. Further, alkyl is C₁～C₆Alkyl of alkoxy C₁～C₆Alkoxy and haloalkyl of (A) are halogeno C₁～C₆Alkyl group of (1).

R^4a、R^4bAnd R⁶Each independently selected from one of hydrogen, alkyl, X-substituted alkyl, cycloalkyl, X-substituted cycloalkyl, heterocyclyl, X-substituted heterocyclyl, aryl, X-substituted aryl, heteroaryl and X-substituted heteroaryl, wherein X is selected from at least one of alkyl, halogen, hydroxyl, amino, mercapto, alkoxy, alkylamino, cycloalkyl, heterocyclyl, carboxyl and carboxylate; alternatively, the first and second electrodes may be,

R^4aand R^4bForm a 4-to 8-membered ring M with the nitrogen atom to which it is attached₁Wherein M is₁Is a substituted heterocyclic group or an unsubstituted heterocyclic group, M₁Containing at least one of N, O or S (O)_mA hetero atom, and M₁The substituent(s) is selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

R³And R⁵With NR^4aR^4bAnd OR⁶One of them is connected and combined to form 4-8 membered ring M₂，M₂Is a substituted heterocyclic group or an unsubstituted heterocyclic group, M₂Containing at least one of N, O or S (O)_mA hetero atom, and M₂The substituent(s) is selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

Further, R¹The structural general formula is as follows:

wherein, X₂₀May be selected from the group consisting of a hydrogen atom, -CH₂CH₂CH₂OH、-CH₂C(CH₃)₂OH、-CH₂CH₂CH₂NHCH₃and-CH₂CH₂CH₂N(CH₃)₂One of (1), X₂₁May be selected from hydrogen atom, halogen and-CH₃One kind of (1).

Further, R¹One selected from the following structural formulas:

wherein the wavy line represents a bond to the nitrogen-containing heterocyclic compound.

Preferably, R²The structural general formula of the substituent group is

Wherein, X₁₁、X₁₂And X₁₃Each independently selected from one of H, hydroxyl and alkoxy, and X₁₁、X₁₂And X₁₃Not all are hydrogen. Further, R²The substituents being selected from

One kind of (1).

Further, the structural general formula of the nitrogen-containing heterocyclic compound is as follows:

in the general formula (II), R¹The definitions of (a) and their preferred forms are in accordance with the above.

In the general formula (II), X₁₁、X₁₂And X₁₃Each independently is a hydrogen atom, a hydroxyl group or an alkoxy group, and X₁₁、X₁₂And X₁₃Not all are hydrogen. Preferably, X₁₁、X₁₂And X₁₃Comprising at least one alkoxy group. Preferably, said X₁₁、X₁₂And X₁₃Each independently is a hydrogen atom or C₁-C₆Alkoxy radical, and X₁₁、X₁₂And X₁₃In at least one C₁-C6 alkoxy.

In some preferred embodiments, the nitrogen-containing heterocyclic compound has the structure

Preferably, the nitrogen-containing heterocyclic compound is selected from one of the following structural formulas:

the nitrogen-containing heterocyclic compound of the above embodiment has at least the following advantages:

(1) on one hand, the nitrogen-containing heterocyclic compound is combined with a tubulin colchicine binding site to promote microtubule depolymerization and inhibit tumor cell proliferation; on the other hand, the protein kinase can inhibit the activity of the kinase by combining with an ATP binding site of the protein kinase, and simultaneously acts on a plurality of targets of the kinase and the tubulin, thereby realizing the simultaneous inhibition of the proliferation and survival of tumor cells by multiple channels and having stronger antitumor activity. Compared with a single-target antitumor drug, the nitrogen-containing heterocyclic compound can simultaneously act on a plurality of targets to generate a synergistic interaction effect, so that the curative effect is improved, and the occurrence of drug resistance can be reduced to a certain extent.

(2) The nitrogen-containing heterocyclic compound has the advantages of novel structure, easier chemical synthesis and lower preparation cost. Compared with medicines aiming at tubulin such as paclitaxel and the like, the medicine has better water solubility and good drug resistance, and is still effective on paclitaxel drug-resistant tumor cells.

The present invention also provides a pharmaceutical composition of an embodiment, wherein an active ingredient of the pharmaceutical composition comprises the nitrogen-containing heterocyclic compound of the above embodiment or a pharmaceutically acceptable salt thereof. Specifically, the pharmaceutical composition can also comprise pharmaceutical excipients and the like commonly used in the field.

The present invention also provides an embodiment of a tubulin inhibitor or a protein kinase inhibitor, comprising the nitrogen-containing heterocyclic compound of the above embodiments or a pharmaceutically acceptable salt thereof.

The present invention also provides a use of the nitrogen-containing heterocyclic compound of an embodiment or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating cancer.

Preferably, the medicament for treating cancer is a medicament for treating leukemia, a medicament for treating rectal cancer, a medicament for treating colon cancer, a medicament for treating lung cancer, a medicament for treating liver cancer, a medicament for treating ovarian cancer, a medicament for treating pancreatic cancer, a medicament for treating prostate cancer, a medicament for treating breast cancer or a medicament for treating cervical cancer.

The invention also provides application of the nitrogen-containing heterocyclic compound or the pharmaceutically acceptable salt thereof in preparation of a protein kinase inhibitor.

Preferably, the protein kinase is selected from at least one of BCR-ABL (Nature Reviews Drug Discovery,2002,1,493), FLT3 (leukamia, 2019,33,299), JNK (Oncotarget,2016,7,27021), MEK5(Biochim biophysis acta.2012,1825,37), STK16(int.j.mol.sci.2019,20,1760) and BMPR (Biochemical Society Transactions,2017,45, 223).

The invention also provides an application of the nitrogen-containing heterocyclic compound or the pharmaceutically acceptable salt thereof in preparing a tubulin inhibitor.

The invention also provides application of the nitrogen-containing heterocyclic compound or the pharmaceutically acceptable salt thereof in preparing a multi-target medicament, wherein the multi-target medicament has double inhibitory activity of tubulin and protein kinase.

The present invention also provides a method for preparing a nitrogen-containing heterocyclic compound according to an embodiment, which is a method for preparing a nitrogen-containing heterocyclic compound according to the above embodiment, including the steps of:

step S110: mixing compound A, R¹B(OH)₂Reacting the palladium catalyst and an alkaline reagent to prepare a compound B.

Wherein the structural formula of the compound A is

The structural formula of the compound B is

Y and Z are respectively and independently selected from one of-CH and-N.

In one embodiment, the palladium catalyst is PdCl₂(dppf) i.e. (1,1' -bis (diphenylphosphino) ferrocene palladium chloride). The alkaline agent is sodium carbonate.

Specifically, compound A, R¹B(OH)₂In the step of reacting the palladium catalyst and the alkaline reagent, a degassed dioxane aqueous solution is further added as a solvent.

Further, in step S110, the reaction temperature is 80-100 ℃ and the reaction time is 1-2 h. Preferably, step S110 is performed in a microwave reactor.

In a specific example, step S110 includes: under an atmosphere of protective gas, compound A, R¹B(OH)₂Mixing the palladium catalyst and the alkaline reagent with the degassed dioxane aqueous solution, and reacting for 1-2 h at 80-100 ℃ in a microwave reactor to prepare a compound B.

Step S120: reacting the compound B with an acidic reagent to prepare a nitrogen-containing heterocyclic compound, wherein the structural formula of the nitrogen-containing heterocyclic compound is shown in the specification

Wherein R is¹And R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl.

When R is¹And R²Is substituted aryl or substitutedWhen the heteroaryl group of (A) is present, R¹And R²Each of the substituents of (A) is independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mOne of heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonylamino group, N-sulfonylamino group, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group and N-acylamino group of hetero atom; m is 0,1 or 2.

In one embodiment, the acidic agent is hydrochloric acid.

Specifically, in the step of reacting the compound B with an acidic reagent, ethanol is further added as a solvent to dissolve the compound B. Further, in one embodiment, in the step of reacting the compound B with the acidic reagent, the reaction temperature is 80 ℃ and the reaction time is 12 hours.

The step of reacting compound B with an acidic reagent may be followed by a purification step. In one embodiment, the step of purifying comprises: concentrating the reaction system under reduced pressure, separating liquid phase, and freeze drying.

In a specific example, step S120 includes: dissolving the compound B in ethanol, adding an acidic reagent, reacting at 80 ℃ for 12h, and after the reaction is finished, sequentially carrying out reduced pressure concentration, liquid phase separation and freeze drying to prepare the nitrogen-containing heterocyclic compound.

Further, the method for preparing the nitrogen-containing heterocyclic compound further includes step S130: compound a was prepared.

When Y and Z are both-CH, step S130 includes step S132 and step S134, which are as follows:

step S132: compound C was reacted with NaH, SEMCl to prepare compound D.

Wherein the structural formula of the compound C is

The structural formula of the compound D is

Specifically, in step S132, DMF was also added to dissolve compound C. In a specific example, step S132 is: dissolving the compound C in DMF, adding NaH at the temperature of 0 ℃, stirring for 10min, then dripping SEMCl, and continuously stirring for reaction at the room temperature. After the reaction is finished, adding water to quench the reaction, extracting the reaction product by using ethyl acetate, drying an organic layer by using anhydrous sodium sulfate, filtering, concentrating, and separating by using column chromatography to prepare the compound D.

Step S134: mixing compound D, R²B(OH)₂Reacting the palladium catalyst and an alkaline reagent to prepare the compound A.

Specifically, compound D, R¹B(OH)₂In the step of reacting the palladium catalyst and the alkaline reagent, a degassed dioxane aqueous solution is further added as a solvent.

Further, in step S134, the reaction temperature is 80-100 ℃ and the reaction time is 1-2 h. Preferably, step S134 is performed in a microwave reactor.

In a specific example, step S134 includes: under an atmosphere of protective gas, compound D, R¹B(OH)₂Mixing the palladium catalyst and the alkaline reagent with the degassed dioxane aqueous solution, and reacting for 1-2 h at 80-100 ℃ in a microwave reactor to prepare a compound B.

Further, step S134 further includes a purification step. The purification steps are specifically as follows: after the reaction, the reaction product is filtered, extracted by ethyl acetate, and an organic layer is dried by anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to prepare a compound D.

When Y is-CH and Z is-N, or Y is-N and Z is-CN, step S130 includes step S131, step S133, step S135 and step S137, which are as follows:

step S131: compound E is reacted with TMPZnCl. LiC to prepare compound F.

Wherein the structural formulas of the compound E and the compound F are respectively

Specifically, in the step of reacting compound E with TMPZnCl · LiC, THF is also added as a solvent. In one embodiment, the step of reacting compound E with TMPZnCl · LiC is performed at room temperature.

Step S133: reacting compounds F and R²COCl and CuCN.2LiC to prepare a compound G.

Wherein the structural formula of the compound G is

Step S135: reacting compounds G and N₂H₄·H₂O、CH₃And (4) OH reaction to prepare a compound H.

Wherein the structural formula of the compound H is

Step S137: compound a was prepared by reacting compound H with NaH, SEMCl.

Specifically, in step S137, DMF was also added to dissolve compound H. In a specific example, step S137 is: dissolving the compound H in DMF, adding NaH at the temperature of 0 ℃, stirring for 10min, then dripping SEMCl, and continuously stirring for reaction at the room temperature. After the reaction is finished, adding water to quench the reaction, extracting the reaction product by using ethyl acetate, drying an organic layer by using anhydrous sodium sulfate, filtering, concentrating, and separating by using column chromatography to prepare the compound H.

In one embodiment, Y and Z are both-CH and the nitrogen-containing heterocyclic compound is synthesized as follows:

in another embodiment, Y is-CH and Z is-N, and the nitrogen-containing heterocyclic compound is synthesized as follows:

in another embodiment, Y is-N, Z is-CH, and the nitrogen-containing heterocyclic compound is synthesized as follows:

the preparation method of the nitrogen-containing heterocyclic compound at least has the following advantages:

(1) the preparation method of the nitrogen-containing heterocyclic compound has the advantages of simple process, easy synthesis and low preparation cost.

(2) The preparation method of the nitrogen-containing heterocyclic compound can prepare the compound which acts on multiple targets of kinase and tubulin and improves the antitumor activity. On one hand, the compound is combined with a tubulin colchicine binding site to promote microtubule depolymerization and inhibit tumor cell proliferation; on the other hand, the protein kinase can inhibit the activity of the kinase by combining with an ATP binding site of the protein kinase, and provides a new idea for the synthesis of antitumor compounds.

The following are specific examples:

the present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

The structural formula of the compound is determined by Nuclear Magnetic Resonance (NMR) and/or liquid mass (LC-MS). NMR was measured using a Bruker 400MHz NMR spectrometer with a shift (. delta.) of 10^-6The unit of (ppm) is given, and the solvent for determination is deuterated dimethyl sulfoxide (DMSO-d)₆) Or deuterated chloroform (CDCl)₃) Internal standard is methylsilane (TMS). MS was determined using waters tandem quadrupole mass spectrometers. Liquid phase separation and purification by GilsAnd (3) preparing the high-performance liquid phase instrument by using the semi-on method. IC50 was determined using a Bioteck plate reader. The microwave reaction was carried out on a Biotage microwave reactor. The starting materials used in the examples of the present invention may be synthesized by or according to methods known in the art, or may be purchased from chemical reagents companies such as Biden, Annagei and Leyan. In the examples, unless otherwise specified, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere.

Experimental parameters not described in the following specific examples are preferably referred to the guidelines given in the present application, and may be referred to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by the manufacturer.

Example 1

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

(1)

compound 1(2g,6.19mmol) was dissolved in DMF, cooled to 0 ℃ in an ice bath, NaH (0.5g,12.39mmol) was added slowly, and after stirring for 10 minutes, SEMCl (2mL,10.53mmol) was added dropwise, and the reaction was stirred at room temperature. After the reaction is finished, water quenching is added for reaction, Ethyl Acetate (EA) is used for extraction, an organic layer is dried by anhydrous sodium sulfate, filtered, concentrated and separated by column chromatography to obtain light yellow solid 2g, and the yield is 71.4%, namely the compound 2. The structural characterization of compound 2 is shown below: LC-MS: m/z 454[ M + H]⁺.¹H NMR(400MHz,CDCl₃)δ8.62(d,J＝2Hz,1H),7.99(d,J＝2Hz,1H),5.83(S,2H),3.66(t,J＝7.2Hz,2H),0.95(t,J＝7.2Hz,2H),-0.03(s,9H).¹³C NMR(100MHz,CDCl₃)δ151.3,149.5,132.5,122.1,113.9,91.6,75.5,67.3,17.7,-1.5.

(2)

Compound 2(1.8g) Compound 3(0.924g), PdCl₂(dppf)(288mg)、Na₂CO₃(1.26g) and a degassed dioxane-water solution (v/v 10:1,17.6mL) were sequentially charged into a reaction flask, replaced with argon, and then placed in a microwave reactor for reaction at 80 ℃ for 2 h. After the reaction is finished, filtering, EA extracting, drying the organic layer by using anhydrous sodium sulfate, filtering, concentrating, and carrying out column chromatography separation to obtain 1.3g of yellow solid, wherein the yield is 66.3%, and the compound 4 is obtained. The structural characterization of compound 4 is shown below: LC-MS: m/z 494[ M + H]⁺.¹H NMR(400MHz,CDCl₃)δ8.60(d,J＝2Hz,1H),8.39(d,J＝2Hz,1H),7.10(s,2H),5.83(S,2H),3.97(s,6H),3.91(s,3H),3.70(t,J＝8.0Hz,2H),0.95(t,J＝8.0Hz,2H),-0.04(s,9H).¹³C NMR(100MHz,CDCl₃)δ155.2,151.9,151.4,144.9,140.4,133.6,129.3,116.7,114.8,106.1,76.7,68.5,62.4,61.8,57.9,19.2,0.0.

(3)

Mixing Compound 4(100mg), Compound 5(34g), PdCl₂(dppf)(15mg)、Na₂CO₃(64mg) and a degassed dioxane-water solution (v/v 10:1,2.2mL) were sequentially charged into a reaction flask, replaced with argon, and then placed in a microwave reactor at 100 ℃ for reaction for 1.5 h. After the reaction is finished, filtering and concentrating to obtain a crude product, namely the compound 6, which is directly used for the next reaction. The structure of compound 6 is characterized as follows: LC-MS: m/z 521[ M + H]⁺.

(4)

Compound 6 was dissolved in ethanol, an equal volume of 2N HCl was added and the reaction was heated at 80 ℃ overnight. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze-drying to obtain a yellow target compound 7, namely the nitrogenous heterocyclic compound of the implementation. The structural characterization of compound 7 is shown below: LC-MS: m/z 391[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ8.82(d,J＝2Hz,1H),8.56(d,J＝2Hz,1H),7.46(m,2H),7.32(m,1H),7.25(s,2H),3.92(s,6H),3.74(s,3H),2.28(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ158.9,158.6,153.9,152.9,148.5,143.7,138.3,137.1,132.0,129.8,129.1,127.9,127.2,126.9,122.6,118.6,112.6,104.8,60.6,56.5,17.2.

Example 2

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

(1)

compound 6(100mg, 0.192mmol) was dissolved in DMF (3mL) and K was added₂CO₃(79mg, 0.576mmol), and compound 8(72mg, 0.576mmol) was added portionwise at an elevated temperature to 80 ℃ for reaction overnight. After the reaction is finished, adding water to quench the reaction, extracting by EA, filtering, and concentrating to obtain a crude product, namely the compound 9 which is directly used for the next reaction. The structure of the crude product is characterized as follows: LC-MS: m/z 565[ M + H]⁺.

(2)

Compound 9 was dissolved in ethanol (6mL), 2N HCl (6mL) was added, and the reaction was allowed to proceed overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze-drying to obtain a yellow target compound 10, namely the nitrogenous heterocyclic compound of the implementation. The structural characterization of compound 10 is shown below: LC-MS: m/z 435[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ8.85(d,J＝2Hz,1H),8.60(d,J＝2Hz,1H),7.28(s,2H),7.21(m,3H),3.92(s,3H),3.74(s,3H),3.66(t,J＝5.6Hz,2H),3.35(t,J＝5.6Hz,2H),2.23(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ159.4,159.0,158.6,158.3,153.8,152.9,148.7,143.5,138.2,137.2,131.6,130.7,129.2,128.0,124.3,118.7,112.6,104.6,60.6,59.0,56.4,47.9,17.4.

Example 3

Nitrogen-containing of this exampleThe structural formula of the heterocyclic compound is:

the preparation process comprises the following steps:

the synthesis of compound 12 from compound 4 and compound 11 is performed by the method of compound 6 in example 1, and details are not repeated herein, and the obtained product is used in the next reaction without purification. The structure of compound 12 is characterized as follows: LC-MS: m/z 507[ M + H]⁺.

Dissolving the compound 12 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 13, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 13 is shown below: LC-MS: m/z 377[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.9(br,1H),8.83(d,J＝2Hz,1H),8.58(d,J＝2Hz,1H),7.43(m,3H),7.26(s,2H),7.04(d,J＝7.6Hz,1H),3.92(s,6H),3.74(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ158.9,153.9,153.0,148.6,143.8,139.6,138.3,130.6,129.9,129.5,129.1,128.2,127.1,121.8,118.4,117.9,112.6,104.8,60.6,56.6.

Example 4

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 15 from compound 4 and compound 14 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. Compound (I)15 are characterized as follows: LC-MS: m/z 508[ M + H ]]⁺.

Dissolving the compound 15 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 16, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 16 is shown below: LC-MS: m/z 378[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ14.11(s,1H),8.97(d,J＝2Hz,1H),8.82(d,J＝2Hz,1H),8.07(d,J＝6.8Hz,1H),8.01(br,1H),7.45(dd,J＝6.8Hz,J＝1.6Hz,1H),7.34(m,1H),7.26(s,2H),3.92(s,6H),3.75(s,3H).

Example 5

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 18 from compound 4 and compound 17 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 15 is characterized as follows: LC-MS: m/z 532[ M + H]⁺.

Dissolving the compound 15 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 19, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 19 is shown below: LC-MS: m/z 402[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ8.91(d,J＝2Hz,1H),8.65(d,J＝2Hz,1H),8.19(br,1H),8.15(br,1H),7.82(dd,J＝8.4Hz,J＝1.6Hz,1H),7.68(d,J＝8.4Hz,1H),7.28(s,2H),3.92(s,6H),3.74(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ153.8,152.7,149.2,143.6,139.9,138.2,134.4,131.1,130.9,129.2,128.2,126.7,124.1,119.6,112.6,111.3,104.8,60.6,56.6.

Example 6

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 21 from compound 4 and compound 20 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 21 is characterized as follows: LC-MS: m/z 532[ M + H]⁺.

Dissolving the compound 21 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 22, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 22 is shown below: LC-MS: m/z 402[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.9(s,1H),13.3(s,1H),8.93(d,J＝2.0Hz,1H),8.74(d,J＝2.0Hz,1H),8.33(s,1H),7.60(d,J＝8.0Hz,1H),7.49(t,J＝7.2Hz,1H),7.39(d,J＝7.2Hz,1H),7.31(s,2H),3.91(s,6H),3.74(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ153.8,152.9,149.5,143.8,141.0,138.2,133.0,131.8,129.8,129.3,129.1,126.9,121.9,120.7,112.6,110.0,104.7,60.6,56.4.

Example 7

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 24 from compound 4 and compound 23 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 24 is characterized as follows: LC-MS: m/z 508[ M + H ]]⁺.

Dissolving the compound 24 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 25, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 25 is shown below: LC-MS: m/z 378[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ8.81(d,J＝2.0Hz,1H),8.56(d,J＝2.0Hz,1H),7.31(t,J＝8.0Hz,1H),7.24(m,3H),6.82(m,1H),3.92(s,6H),3.74(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ158.4,153.8,152.9,148.7,143.6,139.8,138.3,130.6,130.5,129.1,128.2,118.5,115.0,114.6,112.6,104.8,60.6,56.5.

Example 8

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

method for synthesizing compound 27 from compound 4 and compound 26 following the method for synthesizing compound 6 in example 1, no further description is given here, and the obtained product is used in the next reaction without purification. The structure of compound 27 is characterized as follows: LC-MS: m/z 522[ M + H]⁺.

Dissolving the compound 27 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 28, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 28 is shown below: LC-MS: m/z 392[ M + H ]]⁺.¹H NMR(400MHz,DMSO-d₆)δ8.78(d,J＝2.0Hz,1H),8.51(d,J＝2.0Hz,1H),7.25(s,2H),7.15(m,3H),3.92(s,6H),3.74(s,3H),2.18(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ159.0,158.7,156.4,153.9,152.8,148.5,143.5,138.2,137.0,131.7,130.5,129.2,127.6,123.9,118.3,113.8,112.6,104.7,60.6,56.5,16.2.

Example 9

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 30 from compound 4 and compound 29 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 30 is characterized as follows: LC-MS: m/z 552[ M + H ]]⁺.

Dissolving the compound 30 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 31, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 31 is shown below: LC-MS: m/z 422[ M + H ]]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.9(br,1H),8.88(d,J＝2.0Hz,1H),8.65(d,J＝2.0Hz,1H),7.38(m,3H),7.28(s,2H),6.97(m,1H),4.11(t,J＝4.8Hz,2H),3.92(s,6H),3.76(t,J＝4.8Hz,2H),3.74(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ159.8,159.0,158.6,153.8,152.9,148.9,143.7,139.9,138.2,130.6,130.2,129.1,128.6,120.0,114.4,113.7,112.5,104.7,70.1,60.6,60.1,56.5.

Example 10

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

preparation ofThe process is as follows:

the synthesis of compound 33 from compound 4 and compound 32 was carried out according to the synthesis of compound 6 in example 1, and details thereof are omitted, and the obtained product was used in the next reaction without purification. The structure of compound 33 is characterized as follows: LC-MS: m/z 566[ M + H]⁺.

Dissolving the compound 33 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 34, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 34 is shown below: LC-MS: m/z 436[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.8(br,1H),8.88(d,J＝2.0Hz,1H),8.64(d,J＝2.0Hz,1H),7.37(m,1H),7.28(m,4H),4.15(t,J＝5.2Hz,2H),3.92(s,6H),3.76(t,J＝4.8Hz,2H),3.74(s,3H).2.22(s,3H).¹³C NMR(100MHz,DMSO-d₆)δ158.9,158.5,157.8,153.8,152.9,148.9,143.6,138.1,137.4,131.4,130.5,129.2,128.3,125.8,119.5,112.6,111.1,104.6,70.2,60.6,60.2,56.4,16.2.

Example 11

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 36 from compound 4 and compound 32 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 36 is characterized as follows: LC-MS: m/z 566[ M + H]⁺.

Combining the compounds obtained in the last stepSubstance 36 dissolved in EtOH/H at equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 37, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 37 is shown below: LC-MS: m/z 436[ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.9(br,1H),8.88(d,J＝2.0Hz,1H),8.65(d,J＝2.0Hz,1H),7.37(m,3H),7.28(s,2H),6.96(m,1H),4.15(t,J＝6.4Hz,2H),3.92(s,6H),3.74(s,3H),3.59(t,J＝6.4Hz,2H),1.91(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ159.8,153.8,153.0,148.9,143.7,139.9,138.2,130.6,130.2,129.1,128.6,120.0,114.3,113.7,112.5,104.7,65.1,60.6,57.8,56.5,32.6.

Example 12

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

the synthesis of compound 39 from compound 4 and compound 38 was carried out according to the synthesis of compound 6 in example 1, and the details are not repeated herein, and the obtained product was used in the next reaction without purification. The structure of compound 39 is characterized as follows: LC-MS: m/z 580[ M + H ]]⁺.

Dissolving the compound 39 obtained in the previous step in EtOH/H with equal volume ratio₂In O solution, the reaction was carried out overnight at 80 ℃. After the reaction is finished, carrying out reduced pressure concentration, liquid phase separation and purification, and freeze drying to obtain a yellow compound 40, namely the nitrogen-containing heterocyclic compound. The structural characterization of compound 40 is shown below: LC-MS: m/z 450[ M + H ]]⁺.¹H NMR(400MHz,DMSO-d₆)δ13.8(br,1H),8.88(d,J＝2.0Hz,1H),8.63(d,J＝2.0Hz,1H),7.37(m,1H),7.26(s,4H),4.19(t,J＝6.0Hz,2H),3.92(s,6H),3.74(s,3H),3.61(t,J＝6.0Hz,2H),2.20(s,3H),1.91(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ157.7,153.8,152.9,148.8,143.6,138.2,137.4,131.4,130.5,129.2,128.3,125.6,119.3,112.6,110.8,104.6,65.1,60.6,57.9,56.4,32.8,16.2.

Example 13

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation process comprises the following steps:

compound 41(2.0g,1eq) was reacted with TMPZnC. LiCl (21.2mL, 1mol/L,2.5eq) at room temperature for 1 h. CuCl2 LiCl (12.6mL, 1mol/L,1.5eq) and 3,4, 5-trimethoxybenzoyl chloride (3.4g,1.7eq) were added at-78 deg.C, and NH was used after completion of the reaction₄Cl/NH₃·H₂Quenching with O (25% -28%) 9/1, extraction with EA, drying over anhydrous sodium sulfate, and purification with PE/EA 6/1 silica gel column to give compound 44(1.98g, 54.5% yield). The structure of compound 44 is characterized as follows: LC-MS: m/z 433[ M + H]⁺.¹H NMR(400MHz,CDCl₃)δ10.52(s,1H),8.57(s,1H),7.74(s,2H),4.01(s,6H),3.93(s,3H).

Dissolving compound 44(1.98g, 1.0eq) in methanol (46mL), adding excess hydrazine hydrate, heating and refluxing, after the reaction is complete, returning to room temperature, adding DCM until the yellow solid in the reaction solution is completely dissolved, washing the organic phase with saturated NaCl water, and anhydrous Na₂SO₄After drying and spin-drying of the solvent, the residue was recrystallized from methanol, filtered and the methanol was removed in vacuo to yield compound 45 as a yellow solid (1.04g, 73.2% yield). The structure of compound 45 is characterized as follows: LC-MS: m/z 366[ M + H]⁺.¹H NMR(400MHz,CDCl₃)δ8.59(s,1H),7.79(s,2H),5.90(s,2H),4.04(s,6H),3.96(s,3H),3.83–3.36(m,2H),1.04–0.86(m,2H),-0.00(s,9H).¹³C NMR(100MHz,CDCl₃)δ154.96,146.21,144.91,143.68,140.50,136.56,134.02,127.73,105.56,68.80,62.38,57.66,19.08,1.39.

Reacting compound 45 (1)41g, 1.0eq) was dissolved in ultra dry DMF (18mL), 60% NaH (0.31g, 2.0eq) was added at 0 ℃ and stirred for 10min, then SEMCl (1.22mL, 1.7eq) was added and reacted at room temperature, the reaction was completed in about 2.5h, the reaction was quenched by slowly dropping clean water at 0 ℃, the aqueous phase was extracted a few times with EA, the organic phase was washed with saturated NaCl water, dried over anhydrous sodium sulfate and purified with PE/EA 5/1 silica gel column to obtain compound 46 as a yellow solid (1.75g, 91.6% yield). The structure of compound 46 is characterized as follows: LC-MS: m/z 495[ M + H]⁺.¹H NMR(400MHz,CDCl₃)δ8.95(s,1H),7.98(s,2H),7.46(d,J＝6.5Hz,2H),7.22(d,J＝8.0Hz,1H),5.90(s,2H),4.04(s,6H),3.94(s,3H),3.76–3.70(m,2H),2.26(s,3H),1.04–0.91(m,2H),-0.03(s,9H).¹³C NMR(100MHz,CDCl₃)δ153.61,149.47,145.41,144.25,142.89,141.13,136.03,132.23,131.36,127.46,124.23,117.40,113.25,104.40,67.29,61.16,56.34,17.84,17.44,-1.29.

Mixing 46(500mg, 1.0eq), 47(278mg, 1.7eq), Na₂CO₃(321mg, 3.00eq) and PdCl₂(dppf) (74mg, 0.10eq) was dissolved in 1, 4-dioxane (6ml) and H was added₂O (0.6mL), microwave reacted at 100 ℃ for 2h, and after completion of the reaction, the solid in the reaction solution was filtered to retain the liquid, and the organic phase was washed with saturated NaCl water, dried over anhydrous sodium sulfate, and purified by PE/EA-2/1 silica gel column to obtain compound 48(493mg, yield 91.8%) as a yellow solid. The structure of compound 48 is characterized as follows: LC-MS: m/z 533[ M + H ]]⁺.¹H NMR(400MHz,CDCl₃)δ8.90(s,1H),8.01–7.97(m,2H),7.95(s,2H),6.98(d,J＝8.8Hz,2H),6.54(s,1H),5.90(s,2H),4.00(s,6H),3.94(s,3H),3.76–3.71(m,2H),1.01–0.95(m,2H),-0.04(s,9H).¹³C NMR(101MHz,CDCl₃)δ157.75,153.52,149.19,143.89,142.77,140.67,138.68,132.11,129.38,128.53,127.42,116.31,116.29,104.33,75.57,67.35,61.16,56.27,17.82,-1.33.

Dissolving a compound 48(200mg) in 1, 4-dioxane (3mL), adding 4N HCl-dioxane (3mL), directly spin-drying after complete reaction, and performing high performance liquid phase preparation and separation to obtain a yellow solid compound 49, namely the nitrogenous heterocyclic compound of the embodiment. Knot of Compound 49The structure is characterized as follows: LC-MS: m/z 403[ M + H]⁺.¹H NMR(400MHz,DMSO)δ14.15(s,1H),9.70(s,1H),9.19(s,1H),7.90(s,2H),7.70–7.65(m,2H),7.37(t,J＝7.8Hz,1H),6.92(dd,J＝8.0,2.4Hz,1H),3.95(s,6H),3.76(s,3H).¹³C NMR(101MHz,DMSO)δ158.07,153.25,147.68,144.68,141.41,141.32,138.01,137.90,130.17,130.10,127.56,117.59,113.44,103.42,60.17,55.85.

Example 14

The structural formula of the nitrogen-containing heterocyclic compound of the embodiment is as follows:

the preparation method adopts the method of example 13, and the preparation process is as follows:

biological evaluation

(1) Tubulin inhibitory Activity assay

The tubulin inhibition activity was measured using a kit from cytosketon, and the experimental method is briefly as follows: preparing a compound with the final concentration of 3 mu M, adding the compound into a 96-well plate, wherein each well is 10 mu L, each concentration is provided with 3 multiple wells, dissolving tubulin into a prepared tubulin polymerization buffer solution (mainly containing GTP and glycerol according to the specification of a kit), adding 100 mu L of the compound into each well along the inner wall of the 96-well plate, quickly putting the compound into an enzyme labeling instrument to read the value, reading 1 time per minute and continuously reading 61 times. The results of the experiment are shown in FIG. 1.

And (4) experimental conclusion: the nitrogen-containing heterocyclic compound prepared by the invention has obvious inhibitory activity on tubulin aggregation.

(2) Protein kinase inhibitory Activity assay

The experimental method comprises the following steps: screening for kinase Activity Using Eurofins discover X

The screening is carried out by adopting a technical platform or an ADP-Glo kinase activity screening method of Promega companyThe method can be used for testing, and the specific method can refer to the kit instruction.

Taking FLT3 kinase inhibitory activity test as an example, 1 μ L of test compound, positive control drug (Giltertinib) or buffer solution with different concentrations is added into a 384-well plate, 2 μ L of kinase is added, 2 μ L of buffer solution is added into a blank control group, the mixture is mixed uniformly, after incubation for 10min at room temperature, 2 μ L of ATP/substrate (MBP protein) mixed solution is added into each well, the mixture is mixed uniformly, and incubation is carried out for 2h at room temperature. Adding 5 mu LADP-Glo reagent into each hole, mixing uniformly, incubating at room temperature for 40min, adding 10 mu L kinase detection reagent into each hole, mixing uniformly, incubating at room temperature for 30min, detecting chemiluminescence value (RLU) of each hole by an enzyme-labeling instrument, and calculating the kinase inhibition activity of the compound.

Calculating the formula: % inhibition ═ RLU (compound) -RLU (blank))/(RLU (buffer group) -RLU (blank) × 100%.

The results of the experiment are shown in tables 1 and 2 below:

TABLE 1 inhibitory Activity of Compounds on FLT3 kinase

TABLE 2 kinase inhibitory Activity of Compound 10

And (4) experimental conclusion: the nitrogen-containing heterocyclic compound prepared by the invention has obvious inhibitory activity on a plurality of protein kinases.

(3) Cell proliferation inhibition assay

The following experiments were conducted to determine the proliferation inhibitory activity of the compounds of the present invention on tumor cells under in vitro conditions. The inhibitory activity of a compound can be expressed as an IC50 value.

The experimental protocol is briefly described as follows: tumor cells were suspended in a medium containing 10% FBS at an appropriate cell density (e.g., 15000-50000 cells/mL medium), seeded on a 96-well plate, and cultured overnight in a cell culture chamber at 37 ℃ under 5% carbon dioxide. Test compounds were first dissolved in DMSO, then diluted to the concentration required for the experiment (typically 9 concentration points were set for each compound) in FBS-free medium, added to a 96-well plate, and incubated at 37 ℃ in 5% carbon dioxide for an additional 48-72 hours. Thereafter, the inhibitory activity of the compounds on cell proliferation was determined by the CCK8 method. IC50 values for compounds were calculated from the inhibition values of test compounds on cell proliferation at different concentrations, and IC50 values were calculated using GraphPadprism software.

The results of the experiment are shown in table 3 below:

TABLE 3

Examples	Compound ID	HCT116	K562	A549
					Example 1	7	++++	++++	++++
Example 2	10	++++	++++	++++
					Example 3	13	++++	++++	++++
Example 4	16	++++	++++	++
					Example 5	19	++++	++++	+++
Example 6	22	++++	++++	++++
					Example 7	25	++++	++++	++++
Example 8	28	++	++	++
					Example 9	31	+	++	+
Example 10	34	++++	+++	+++
					Example 11	37	+	++	+
Example 12	40	+++	+++	++
					Example 13	49	++++	++++	+++
Example 14	57	++++	++++	++++

In the above table, + +++ denotes an IC50<100 nM; + + + + denotes an IC50 of 100nM to 500 nM; + denotes an IC50 of 500nM to 1000 nM; + denotes IC50>1000 nM.

And (4) conclusion: the nitrogen-containing heterocyclic compound prepared by the invention has obvious inhibition activity on tumor cell proliferation.

(4) Drug resistance test

Firstly, a paclitaxel-resistant non-small cell lung cancer A549 model is constructed in vitro by adopting a medicament continuous contact concentration increasing induction method. The inhibitory activity of the compounds and paclitaxel on cell proliferation was then determined by the CCK8 method.

The results of the experiment are shown in table 4 below:

TABLE 4

And (4) conclusion: the taxol cannot play a good inhibition role on the proliferation of tumor cells, the tumor cells have drug resistance to the taxol, but have no drug resistance to the nitrogen-containing heterocyclic compound prepared by the invention, and the nitrogen-containing heterocyclic compound prepared by the invention has a remarkable inhibition activity on the taxol-resistant tumor cells.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only describe several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the present invention as set forth in the appended claims.

Claims (10)

1. A nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, wherein the nitrogen-containing heterocyclic compound has a structural formula as shown below:

wherein Y and Z are independently selected from-CH and-N;

R¹and R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl;

when said R is¹Or said R²When substituted aryl or substituted heteroaryl, the corresponding substituents are each independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mHetero-atom heterocyclyl, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino and N-acylamino, and m is 0,1 or 2.

2. The nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is¹Is selected from

One of (1);

wherein R is³、R⁵、R⁷And R⁸Each independently selected from hydrogen, alkyl, halogen, alkoxy, haloalkyl, cyano, amino, substitutedOne of an amino group and a hydroxyl group of (a);

R^4a、R^4band R⁶Each independently selected from one of hydrogen, alkyl, X-substituted alkyl, cycloalkyl, X-substituted cycloalkyl, heterocyclyl, X-substituted heterocyclyl, aryl, X-substituted aryl, heteroaryl, and X-substituted heteroaryl, wherein X is selected from at least one of alkyl, halogen, hydroxy, amino, mercapto, alkoxy, alkylamino, cycloalkyl, heterocyclyl, carboxy, and carboxylate;

or R^4aAnd R^4bForm a 4-to 8-membered ring M with the nitrogen atom to which it is attached₁Said M is₁Is a substituted heterocyclic group or an unsubstituted heterocyclic group, said M₁Containing at least one of N, O or S (O)_mAnd said M is₁The substituent(s) is selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

3. The nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 2, wherein R is³And R⁵With NR^4aR^4bAnd OR⁶One of them is connected and combined to form 4-8 membered ring M₂Said M is₂Is a substituted heterocyclic group or an unsubstituted heterocyclic group, said M₂Containing at least one of N, O or S (O)_mAnd the substituent of the substituted heterocyclic group formed is at least one selected from the group consisting of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylate, and m is 0,1 or 2.

4. The nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein R is²Is selected from

One kind of (1).

5. The nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the nitrogen-containing heterocyclic compound is selected from one of the following structural formulae:

6. a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the nitrogen-containing heterocyclic compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. A tubulin inhibitor or a protein kinase inhibitor comprising the nitrogen-containing heterocyclic compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

8. Use of the nitrogen-containing heterocyclic compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating cancer or for the preparation of a protein kinase inhibitor or for the preparation of a tubulin inhibitor or for the preparation of a multi-target medicament.

9. The preparation method of the nitrogen-containing heterocyclic compound is characterized by comprising the following steps:

mixing compound A, R¹B(OH)₂Reacting palladium catalyst with alkaline reagent to prepare a compound B, wherein the structural formula of the compound A is shown in the specification

Said combination ofThe structural formula of the compound B is

Reacting the compound B with an acidic reagent to prepare a nitrogen-containing heterocyclic compound, wherein the structural formula of the nitrogen-containing heterocyclic compound is shown in the specification

Wherein Y and Z are respectively and independently selected from one of-CH and-N, R¹And R²Each independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl;

when said R is¹And said R²When it is substituted aryl or substituted heteroaryl, said R¹And said R²Each of the substituents of (A) is independently selected from halogen, alkyl, cycloalkyl, substituted C₁～C₆Alkyl, substituted halogeno C₁～C₆Alkyl, substituted C₁～C₆Alkenyl, substituted C₁～C₆Alkynyl, hydroxy, substituted C₁～C₆Alkoxy, substituted halogeno C₁～C₆Alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, substituted 4-to 8-membered ring containing at least one of N, O or S (O)_mOne of heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonylamino group, N-sulfonylamino group, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group and N-acylamino group of hetero atom; m is 0,1 or 2.

10. The method for producing a nitrogen-containing heterocyclic compound according to claim 9, characterized by further comprising a step of producing the compound a;

when Y and Z are both-CH, the step of preparing said compound A comprises:

reacting compound C with NaH and SEMCl to prepare compound D, wherein the structural formula of the compound C is shown in the specification

The structural formula of the compound D is

Subjecting said compound D, R²B(OH)₂Reacting a palladium catalyst and an alkaline reagent to prepare the compound A; alternatively, the first and second electrodes may be,

one of Y and Z is-CH and the other is-N, and the preparation method of the compound A comprises the following steps:

reacting the compound E with TMPZnCl. LiC to prepare a compound F, wherein the structural formulas of the compound E and the compound F are respectively shown in the specification

Subjecting said compounds F and R²COCl and CuCN.2LiC to prepare a compound G, wherein the structural formula of the compound G is shown in the specification

Reacting said compounds G and N₂H₄·H₂O、CH₃OH reaction to prepare a compound H, wherein the structural formula of the compound H is shown in the specification

Reacting the compound H with NaH and SEMCl to prepare the compound A.

Images